ABSTRACT
Using a yeast two-hybrid screen, we identified human nucleosome assembly protein 1 (hNAP-1) as a protein interacting with the activation domain of the transcriptional activator encoded by papillomaviruses (PVs), the E2 protein. We show that the interaction between E2 and hNAP-1 is direct and not merely mediated by the transcriptional coactivator p300, which is bound by both proteins. Coexpression of hNAP-1 strongly enhances activation by E2, indicating a functional interaction as well. E2 binds to at least two separate domains within hNAP-1, one within the C terminus and an internal domain. The binding of E2 to hNAP-1 is necessary for cooperativity between the factors. Moreover, the N-terminal 91 amino acids are crucial for the transcriptional activity of hNAP-1, since deletion mutants lacking this N-terminal portion fail to cooperate with E2. We provide evidence that hNAP-1, E2, and p300 can form a ternary complex efficient in the activation of transcription. We also show that p53 directly interacts with hNAP-1, indicating that transcriptional activators in addition to PV E2 interact with hNAP-1. These results suggest that the binding of sequence-specific DNA binding proteins to hNAP-1 may be an important step contributing to the activation of transcription.
Subject(s)
DNA-Binding Proteins/metabolism , Nuclear Proteins , Proteins/metabolism , Trans-Activators/metabolism , Transcription, Genetic , Transcriptional Activation , Viral Proteins/metabolism , Acetyltransferases/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Line , DNA-Binding Proteins/genetics , Genes, Reporter , Histone Acetyltransferases , Humans , Macromolecular Substances , Nucleosome Assembly Protein 1 , Protein Binding , Protein Structure, Tertiary , Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , TEA Domain Transcription Factors , Trans-Activators/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Two-Hybrid System Techniques , Viral Proteins/genetics , p300-CBP Transcription FactorsABSTRACT
The E2 proteins regulate papillomavirus (PV) gene expression by sequence-specific DNA binding. However, E2 is also able to activate in the absence of E2 binding sites. We show here that the E2 protein of human PV type 8 (HPV8) can activate the expression of p21(WAF1/CIP1) via promoter-proximal 200 nucleotides, which contain several Sp1 binding sites and no E2 binding sites. HPV8 E2 lacking the activation domain, which is rather conserved among E2 proteins, cooperated with co-expressed Sp1 in stimulation of the p21(WAF1/CIP1) promoter, in contrast to HPV18 E2 lacking the activation domain. We can demonstrate that the internal non-conserved hinge region of HPV8 E2 is sufficient for this functional cooperativity with Sp1. In correlation, the hinge of HPV8 E2 directly binds to Sp1. These results suggest that HPV8 E2 might be able to 'super'-activate Sp1-mediated transcription by a direct interaction via the non-conserved hinge region.
